Linking the conformational landscape to enzymatic function through functional site distant mutations

通过功能位点远距离突变将构象景观与酶功能联系起来

• 批准号: 10543155
• 负责人: Thomas Michael Sabo
• 金额: $ 32.44万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10543155
• 关键词: 2019-nCoV; Active Sites; Affect; Antineoplastic Agents; Architecture; Binding; Calorimetry; Catalysis; Cyclic GMP; DNA; Data; Development; Dissociation; Distant; Enzymes; Equilibrium; Future; Generations; Grant; Guanosine Triphosphate; Guanylate kinase; Human; Kinetics; Laboratories; Ligand Binding; Ligands; Link; Malignant Neoplasms; Measurable; Measures; Methods; Molecular Conformation; Motion; Muramidase; Mutation; NMR Spectroscopy; Nuclear Magnetic Resonance; Nucleosides; Nucleotides; Outcome; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Play; Probability; Production; Proteins; Protocols documentation; RNA; Rationalization; Regulation; Relaxation; Research Personnel; Residual state; Role; SARS coronavirus; SUMO1 gene; Sampling; Scheme; Series; Shapes; Single Nucleotide Polymorphism; Site; Structural Biologist; Structure; Testing; Therapeutic; Thermodynamics; Titrations; Ubiquitin; Variant; Vertebral column; Viral; Work; cancer cell; conformational conversion; design; drug discovery; enthalpy; inhibitor; molecular dynamics; mutant; small molecule inhibitor; therapeutic target; tool

An ongoing debate concerns the role conformational motions, often termed dynamics, play in biomolceular funtion. For enzymes, it so happens that the timescales for large-scale domain motions are similar to the apparent "$$). catalytic rate (𝑘!"# This observation is where the major point of contention has developed: do "$$. In this context, the proposed studies conformational motions directly impact the true rate of catalysis (𝑘!"#) or 𝑘!"# will further explore how the modulation of the conformational landscape can indeed fine-tune "$$ without 𝑘!"# impacting 𝑘!"# and the ground state structure. The genesis of this proposal arises from our work with human guanylate kinase (hGMPK), a potential therapeutic target for treating cancer and perhaps even SARS-CoV-2, which motivated us to solve the first structure of hGMPK with nuclear magnetic resonance (NMR) spectroscopy (PDB: 6NUI). While solving the hGMPK structure, we expressed a series of seven functional site distant (FSD), "$$ when compared to the wild- non-synonymous single nucleotide variants (nsSNVs) of hGMPK that enhance 𝑘!"# type (wt). Intriguingly, the 2D [1H,15N]-HSQC NMR spectra of the wt hGMPK and its nsSNVs suggest that the 𝑎𝑝𝑝) for GMP binding to FSD mutations minimally impact hGMPK’s backbone fold, yet the apparent off-rates (𝑘𝑜𝑓𝑓 wt and the FSD mutant V91M differ by ~3000 s-1. We hypothesize that hGMPK’s activity can be modulated with FSD mutants by reshaping the conformational landscape. Utilizing NMR spectroscopy and isothermal calorimetry, we will test this hypothesis in the following two Specific Aims. In Aim 1, we will quantify the impact of the FSD mutations on the conformational landscape from kinetic and thermodynamic perspectives. The results from this Aim will provide a comprehensive picture as to where within the hGMPK catalytic and binding schemes the FSD mutations have the largest impact on function. For Aim 2, we will deconvolute the contribution transient structures within the conformational landscape play in enzymatic catalysis through experimentally driven ensemble generation. Our protocol will select hGMPK structures from unbiased molecular dynamics (MD) simulations based on residual dipolar couplings and cross-correlated relaxation rates measured with NMR. The ensembles will aid in the identification of the functionally important transient conformations and an assessment of the impact the FSD mutations have on backbone dihedral correlated motions. To our knowledge, this proposal provides the first examples 1.) of experimentally driven, ensemble generation for an enzyme spanning physiologically relevant timescales and 2.) of determining thermodynamic and kinetic parameters with ligand binding to the same exact site on a series of enzyme variants. The impact of this proposal is the direct linkage of the conformational landscape to enzymatic function. Immediate applications for these results include drug discovery, where targeting structures within the conformational landscape rather than the ground state structure will lead to better outcomes, and biomolecular design, where FSD mutations can be implemented to adjust function through manipulation of the conformational landscape.

一场持续的争论涉及构象运动（通常称为动力学）在生物分子中发挥的作用 函数。对于酶来说，大规模域运动的时间尺度与 明显的“$$）。 催化率 (𝑘!"# 这一观察结果是争论的主要焦点：做 “$$。在这方面，拟议的研究 构象运动直接影响催化的真实速率 (𝑘!"#) 或 𝑘!"# 将进一步探索构象景观的调节如何确实可以微调“$$而无需 𝑘！”# 影响𝑘！"#和基态结构。这个提议的起源源于我们与人类的合作 鸟苷酸激酶 (hGMPK)，是治疗癌症甚至 SARS-CoV-2 的潜在治疗靶点， 这促使我们利用核磁共振 (NMR) 光谱解析了 hGMPK 的第一个结构 （PDB：6NUI）。在求解hGMPK结构的同时，我们表达了一系列七个功能位点远距离（FSD）， “与野生相比$$ hGMPK 的非同义单核苷酸变体 (nsSNV) 增强 𝑘！"# 类型（重量）。有趣的是，wt hGMPK 及其 nsSNV 的 2D [1H,15N]-HSQC NMR 谱表明 𝑎𝑝𝑝）用于 GMP 绑定 FSD 突变对 hGMPK 的骨架折叠影响最小，但明显的解离率 (𝑘𝑜𝑓𝑓 wt 和 FSD 突变体 V91M 相差约 3000 s-1。我们假设 hGMPK 的活性可以通过以下因素调节： FSD 突变体通过重塑构象景观。利用核磁共振波谱和等温 量热法，我们将在以下两个具体目标中检验这一假设。在目标 1 中，我们将量化影响 从动力学和热力学角度研究 FSD 突变对构象景观的影响。结果 从此目标将提供 hGMPK 催化和结合方案内的全面信息 FSD 突变对功能的影响最大。对于目标 2，我们将对瞬态贡献进行反卷积 构象景观中的结构通过实验驱动在酶催化中发挥作用 乐团一代。我们的方案将从无偏分子动力学 (MD) 中选择 hGMPK 结构 基于残余偶极耦合和 NMR 测量的互相关弛豫率的模拟。这 整体将有助于识别功能上重要的瞬态构象并进行评估 FSD 突变对骨干二面相关运动的影响。据了解，该提案 提供了第一个例子 1.) 实验驱动的、跨酶的整体生成 生理相关的时间尺度和2.)用配体确定热力学和动力学参数 结合到一系列酶变体上的相同精确位点。该提案的影响是直接联系 构象景观与酶功能的关系。这些结果的直接应用包括药物 发现，其中针对构象景观内的结构而不是基态结构 将带来更好的结果和生物分子设计，其中可以实施 FSD 突变来调整 通过操纵构象景观来发挥功能。